A calcium salt crystal controlling bacterium was isolated from the Antarctic Lake Vanda, which contains a high concentration of this calcium compound, and identified as Bacillus sp. based on its taxonomical characteristics. A calcium carbonate-crystal-growth inhibitor (CCGI) from the Antarctic bacterium, and present in cells, strongly inhibits precipitation from supersaturated calcium carbonate solutions. Therefore, we suggest that this new secretor protein may act as an inhibitor of spontaneous calcium carbonate precipitation from supersaturated pancreatic juice. CCGI from an extract of this strain was purified and characterized. We found that the purified CCGI was a monomer of approximately 7,100 according to gel filtration chromatography (Sephacryl S-100) and SDS-PAGE, and was a heat-stable protein. The protein inhibited the crystal growth of calcium carbonate and also affected the crystal morphology. Most of the crystals showed a rhombohedra crystal morphology in the absence of CCGI. On the other hand, CCGI produced different initial morphologies, i.e., the rounded shape for CCGI. These CCGI peptides may interact with different sites of the growing crystals, preventing further accretion of the mineral, thereby changing the crystal morphology.

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In order to study the freezing process of food materials, a freezing experiment was carried out on an onion piece. We focused on the epidermal cells of onion that were obtained from the first scaly leaves. Further, we discussed the freezing process by examining the changes in viability of these cells and the appearance of protoplasts after exposing them to a low temperature. The cells were maintained at a temperature of -34.5℃ for 0, 1, 2, 4, 8, and 15h. After thawing, the cell viability was calculated from the observations made on protoplasmic streaming for more than 160 cells, under each of these conditions. The results showed that the viability of the fresh onion cells was approximately 100% and this decreased proportionally with the cooling time until it reached almost 0% at 8h. In particular, both a rapid decrease of viability was detected after 2h of cooling, when the cells were passing through the zone of maximum ice crystal formation. This result suggested that the main cause of cell death was freezing. The appearance of cells was different, depending on cooling time. Under the condition of relatively short term cooling such as for 1 to 2h, the transform of dead protoplast was not obviously. These cells seem to be killed by mechanical damage to cell membrane by extracellular freezing; however, if the cells were cooled for more than 4h, they displayed shrinkage and broken protoplast fragments. This implies that the severe mechanical damage occurred after the shrinkage of cells due to severe extracellular freezing.

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Three rice lines were evaluated for vivipary, so called pre-harvest sprouting from the view point of dynamic states of water using nearly isogenic line. At 28 days-post anthesis (DPA) germination rate in NK2, as a pre-harvest resistant line, was similar to nk2, as a preharvest sesitive line, suggesting that there seems to be other genes controlling vivipary except for a single gene. At this stage both coleoptiles and radicles for NK2 and Taichung 65 (TC65) began to elongate, and the organs of TC65 were shortest among three lines. Therefore, it was indicated that rice seeds treated over 28 DPA easily lost their dormancy. The germination tests indicated that the genotypes did not differ in response to ABA in the mature seeds. In addition, there was no influence of ABA on water uptake into non-sprouting endosperms as well as sprouting endosperms of the three rice lines. ABA treatment decreased both T_2s of the both fractions in endosperms of the rice lines compared to those of DW treatment. These results suggested that the water bound with macromolecules appears to remain unchanged by ABA-treatment even in the sprouting phenomenon

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Effects of temperature below ice point on vigor and germination rate, and quality characteristics of rice during four-year storage were investigated in this study. Low temperature maintained dormancy of rice and hence preserved vitality of rice. There was very little deterioration in the quality of rice stored at low temperature. Eating quality of rice stored at temperatures less than 5℃ for four years was the same as that of newly harvested rice. These results indicate that preservation of rough rice quality in farm scale silos for several years is possible by storing rice at an average temperature below 5℃ during storage.

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Larvae of an African chironomid, Polypedilum vanderplanki live in temporal rock pools and they can stand complete desiccation in a cryptobiotic (ametabolic) state. The dehydrated larvae are able to revive within 1hr after rehydration. Here we studied the physicochemical mechanism of the cryptoiosis using FTIR microspectroscopy and DSC. When slowly dehydrated in the laboratory conditions over 48hrs, larvae synthesized a large amount of trehalose i.e. 36μg/individual and successfully went into cryptobiosis. In contrast the larvae quickly desiccated within several hrs produced as much as 3μg/individual of trehalose and never recovered after rehydration. Trehalose has a characteristic IR peak at 992cm^<-1>. We compared intensity distributions of this peak over the whole body followed by two dimensional contour maps for trehalose concentration between slowly and quickly dehydrated larvae. It was revealed that trehalose was almost uniformly distributed in the slow sample, but only little trehalose was detected in the quick sample. DSC measurements demonstrated that trehalose in the slow sample was vitrified with a glass transition temperature of ca. 50℃ (mid point). We obtained recovery rate of the rehydrated larvae after exposuring different temperatures for 5 min or 1h, which was interestingly well correlated with the glass transition behavior as mentioned above. We therefore concluded that vitrification of trehalose accumulated in the whole body was of importance in the successful induction of cryptobiosis in P. vanderplanki larvae.

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We studied super-low temperature preservation of moss (Pogonatum inflexum) protonemata. Pogonatum protonemata could be cultured both in MS and C1 medium. Addition of sucrose to C1 medium enhanced the growth of protonemata significantly, whereas MS medium was not suitable for the rapid growth of protonemata. Protonemata cultured in C1 medium were cryopreserved by the pre-freezing method. When protonemata were cultured in the mdium containing the higher concentrations (5 or 10%) of sugar, survival rates of the preserved cells were higher than that of the cells which was cultured with the lower concentration (1%) of sugar. Preculture of protonemata with abscisic acid was also effective for elevating the survival rate of preserved cells. From the morphological observations, number of round or oval-shaped cells increased when protonemata were cultured with higher concentration of sugar or abscisic acid. Thus, there may be important relation between morphology of protonema cell and its freezing tolerance.

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Cultured rice cells were cryopreserved in liquid nitrogen. Cells pre-cultured with 0.4M sucrose survived cryopreservation by vitrification method. But the rate of survived cells was very low. On the other hand, the higher survival rate was obtained when the cells were cryopreserved by the pre-freezing method. It was important to culture cryopreserved cells on the ammonium ion-free medium for the higher survival rate. The viability of cryopreserved cells that were cultured on ammonium ion-free medium for only initial 8 hours was the same level to that of the cells cultured on ammonium ion-free medium for all the period. This result suggest that freezing injury of rice cells can be recovered rapidly during the initial stage of post-thaw culture when cultured with the condition of ammonium ion-free.

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Anhydrous amorphous raffinose and trehalose were examined their crystallinity in humid conditions by a powder X-ray diffractometer. The raffinose started to crystallize in the condition of 52% relative humidity at 35℃ after 49 hours and it crystallized completely after 304 hours. The trehalose crystallized completely in the same condition after 3 hours. Anhydrous amorphous raffinose is harder to crystallize than anhydrous amorphous trehalose. Glassy preservation of lactate dehydrogenase (LDH) was carried out in the humid conditions using these sugars as excipients. The enzyme in a trehalose system has a higher activity than in a raffinose system.

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In-situ observations of an ice crystal growing or melting in trehalose or sucrose solutions of various concentrations were performed to study the concentration dependence of melting point of the ice crystal in each solution. Melting point of the ice crystal in trehalose solution was identical with that in sucrose solution. The experimental values of melting points agreed with the formula of the molar depression of freezing point that is applicable in low concentration aqueous solutions when free water was expected to be remaining. Above the molality to be estimated that no free water exist, melting point of ice crystal was lower than the formula of the molar depression of freezing point.

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To obtain the basically knowledge of the molecular mobility of low molecular carbohydrate under the glassy state, the enthalpy relaxation time of all gluco-di-saccharides and glycosyltrehaloses were examined through aging experiment by using DSC. The glassy samples were aged at several temperatures of (Tg-10)K to (Tg-40)K. The enthalpy relaxation time at each temperature was obtained by fitting Kohlrausch- Williams- Watts equation to the time course of enthalpy relaxation amount (ΔH). When the temperature dependence of molecular mobility was assumed as Arrhenius process, the activation energy amount (ΔE) could be estimated. In tested gluco-disaccharide, trehalose and isotrehalose were most highest Tg, and neo trehalose was the lowest, and the difference with the trehalose was 46K. Furthermore, the ΔE was larger in order to trehalose, isotrehalose>> laminaribiose, cellobiose> maltose> kojibiose, sophorose, nigerose, isomaltose, gentibiose>> neotrehalose. Tg and ΔE of glycosyltrehaloses were higher than maltooligo-saccharides. These results supported the explanation of availability of glassy trehalose and glycosyltrehalose as lyoprotectant of protein or biosystem under the severe circumstance.

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For understanding physicochemical and biological implications of amphoteric ions, we investigated hydration of glycinebetaine (GB) by obtaining the water sorption isotherm, and freezing behaviour of the aqueous GB solution by the use of differential scanning calorimetry (DSC). It was found, as a result, that GB is very hygroscopic and easily deliquesces. In the DSC cooling trace, the aqueous GB solution did not show an exotherm due to eutectic separation. However, it appeared during the subsequent heating, followed by the melting endotherm at -26℃. The solid-liquid state diagram indicated that Tg'for the freeze-concentrated GB system was very low (ca. -98℃) with the amount of unfrozen water estimated from the Cg'being large (ca. 58wt%). From these results, amphoteric GB ions are considered to be stabilized by hydration, and viscosity of the GB solution might remain rather low as a result of hydration even when it is freeze-concentrated, causing the solution not to be readily vitrified. Characteristics of hydration of GB explained above probably are relevant to its functions in the life process.

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Isotope effect on T_H (homogeneous nucleation temperature) was investigated on aqueous solutions of (CH_3) _4NC1 and DMSO. The T_H difference between the (CH_3) _4NC1-H_2O solution and the (CH_3) _4NC1-D_2O solution is about 4℃ and changes little with increasing solute concentration. It is shown that the isotope effect due to solute is very small even at high solute concentrations whereas that due to solvent is about 5℃ at dilute solute concentrations and decreases a little at high solute concentrations. It seems that the T_H difference between the DMSO-H_2O solution and the DMSO-D_2O solution apparently becomes larger with increasing pressure.

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